活体小鼠微血管血流倒置双光子激光扫描显微镜检测方法的建立

刘皎; 丛馨; 何其华

doi:10.16098/j.issn.0529-1356.2022.02.018

PDF(3744 KB)

解剖学报 ›› 2022, Vol. 53 ›› Issue (2) : 261-265. DOI: 10.16098/j.issn.0529-1356.2022.02.018

技术方法

活体小鼠微血管血流倒置双光子激光扫描显微镜检测方法的建立

刘皎¹丛馨²何其华^1*

作者信息 +

Establishment of in vivo mice inverted two-photon laser scanning microscope microvascular blood flow imaging

LIU Jiao¹ CONG Xin² HE Qi-hua^1*

Author information +

文章历史 +

摘要

目的使用倒置双光子显微镜，建立连续及质量更好的活体微血管成像及血流计算。方法将麻醉和尾静脉注射染料的小鼠放置于37 ℃热板中央，剥离的组织器官放置于打磨自制的低边缘共聚焦小皿内，生理盐水浸没，自制不同重量和大小的圆形金属垫片按压暴露的组织器官。利用上述改进的装置测量孕鼠（n=3）和正常雌鼠（n=3）的肝脏及后肢的微血管血流差异。结果此方法可以实现稳定连续成像，孕鼠比正常雌鼠的肝脏及后肢血流速度更快。结论此方法能对组织器官进行微血管三维立体成像和血流速度检测。

Abstract

Objective To establish a method of in vivo microvascular imaging and blood flow calculation with better continuous imaging quality. Methods Anesthetized mice with dye injection through tail vein were placed in the center of a 37 ℃ hot plate holder. The stripped tissues were placed in a self-made low edge confocal dish and immersed in normal saline. The exposed tissues were pressed with self-made circular metal pads of different weights and sizes. The microvascular blood flow in the liver and hind limb of pregnant mice (n=3) and normal female mice (n=3) were measured by the improved device. Results This method can accomplish stable and continuous imaging. The blood flow velocity of liver and hind limb of pregnant mice were faster than that of normal female mice. Conclusion This method can be used for three-dimensional imaging of microvessels and detection of blood flow velocity in organs.

导出引用

刘皎丛馨何其华. 活体小鼠微血管血流倒置双光子激光扫描显微镜检测方法的建立[J]. 解剖学报. 2022, 53(2): 261-265 https://doi.org/10.16098/j.issn.0529-1356.2022.02.018

LIU Jiao CONG Xin HE Qi-hua. Establishment of in vivo mice inverted two-photon laser scanning microscope microvascular blood flow imaging[J]. Acta Anatomica Sinica. 2022, 53(2): 261-265 https://doi.org/10.16098/j.issn.0529-1356.2022.02.018

中图分类号： R331

参考文献

［1］ Erdener SE, Dalkara T. Small vessels are a big problem in neurodegeneration and neuroprotection ［J］. Front Neurol, 2019, 10: 889.

［2］ Zenclussen AC, Olivieri DN, Dustin ML, et al. In vivo multiphoton microscopy technique to reveal the physiology of the mouse placenta ［J］. Am J Reprod Immunol, 2012, 68(3): 271-278.

［3］ Tennant KA, Brown CE. Diabetes augments in vivo microvascular blood flow dynamics after stroke ［J］. J Neurosci, 2013, 33(49): 19194-19204.

［4］ Helmchen F, Kleinfeld D. Chapter 10. in vivo measurements of blood flow and glial cell function with two-photon laser-scanning microscopy ［J］. Methods Enzymol, 2008, 444: 231-254.

［5］ Rehberg M, Krombach F, Pohl U, et al. Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy ［J］. PLoS One, 2011, 6(11): e28237.

［6］ Rothstein EC, Nauman M, Chesnick S, et al. Multi-photon excitation microscopy in intact animals ［J］. J Microsc, 2006, 222(Pt 1): 58-64.

［7］ Mizuno H, Kikuta J, Ishii M. In vivo live imaging of bone cells ［J］. Histochem Cell Biol, 2018, 149(4): 417-422.

［8］ Ishii T, Ishii M. Intravital two-photon imaging: a versatile tool for dissecting the immune system ［J］. Ann Rheum Dis, 2011, 70(Suppl 1): i113-115.

［9］ de Moraes LV, Tadokoro CE, Gomez-Conde I, et al. Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of plasmodium-infected erythrocytes ［J］. PLoS Pathog, 2013, 9(1): e1003154.

［10］ Tanaka K, Morimoto Y, Toiyama Y, et al. In vivo time-course imaging of tumor angiogenesis in colorectal liver metastases in the same living mice using two-photon laser scanning microscopy ［J］. J Oncol, 2012, 2012: 265487.

［11］ Tanaka K, Morimoto Y, Toiyama Y, et al. Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy ［J］. Microsc Res Tech, 2012, 75(3): 307-315.

［12］ Cong X, Zhang Y, He QH, et al. Endothelial tight junctions are opened in cholinergic-evoked salivation in vivo ［J］. J Dent Res, 2017,96(5):562-570.

［13］ Chen ZhJ, Fan J, Wang YCh, et al. Observing and detecting on blood flow in hepatic sinus in hepatic steatosis mice ［J］. Acta Anatomica Sinica, 2019, 50(5): 645-650. (in Chinese)

陈重九，樊婧，王宇辰，等. 脂肪肝对小鼠肝血窦内血流状况的影响［J］. 解剖学报，2019，50(5): 645-650.

［14］ Fan J, Chen ChJ, Wang YCh, et al. Hemodynamic changes in hepatic sinusoids of hepatic steatosis mice ［J］. World J Gastroenterol, 2019, 25(11): 1355-1365.

［15］ Li Z, Wei XH, Pan CS, et al. QiShenYiQi pills, a compound Chinese medicine, prevented cisplatin induced acute kidney injury via regulating mitochondrial function ［J］. Front Physiol, 2017, 8: 1090.